Measurement of 20-Hydroxyeicosatetraenoic Acid in Human Urine by Gas Chromatography-Mass Spectrometry

Abstract

Arachidonic acid can be metabolized by cytochrome P450 enzymes to a range of compounds that play a central role in the regulation of vascular tone, renal function, and blood pressure (1)(2). In the vasculature, smooth muscle cells produce 20-hydroxyeicosatetraenoic acid (20-HETE) as a major product of CYP450 metabolism. 20-HETE can cause vasoconstriction by inhibition of potassium channels and is thought to contribute to the vasoconstrictor action of hormones such as angiotensin II and endothelin (3)(4). Despite the physiologic importance of CYP450 metabolites of arachidonic acid, very little is known about the regulation of the concentration of 20-HETE in biological fluids or the relationship of these concentrations with physiologic state in healthy individuals. This has in part been attributable to the lack of reliable sensitive and specific assays to measure endogenous concentrations of these compounds. Gas chromatography–mass spectrometry (GCMS) has been used successfully to measure 20-HETE in biological samples. However, available methods rely on one or more thin-layer chromatography steps (5)(6), and for human urine the presence of interfering peaks can be a problem (7). An alternative procedure has recently been reported that uses a sensitive fluorescent HPLC assay (8), although this may lack the specificity of MS.

We have developed a simplified and reliable method for the analysis of urinary 20-HETE and analyzed 20-HETE concentrations in 24-h urine samples from a group of 30 healthy individuals. Our method involves the use of a single solid-phase extraction cartridge containing both reversed-phase and strong anion-exchange packing followed by HPLC separation before derivatization and GCMS analysis. We have found that preparation of the tert -butyldimethylsilyl derivative (tBDMS), as originally used by Prakash et al. (6), gives …

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DOI: 10.1373/clinchem.2003.023259
Measurement of 20-Hydroxyeicosatetraenoic Acid in
Human Urine by Gas Chromatography–Mass Spectrom-
etry, Jennifer Rivera,
1
Natalie Ward,
1
Jonathan Hodgson,
1
Ian
B. Puddey,
1
John R. Falck,
2
and Kevin D. Croft
1*
(
1
School of
Medicine and Pharmacology, University of Western Aus-
tralia and Western Australian Institute for Medical Re-
search, Perth, Western Australia;
2
Biochemistry Depart-
ment, University of Texas Southwestern Medical Center,
Dallas, TX; * address correspondence to this author at:
School of Medicine and Pharmacology, GPO Box X2213,
Perth, Western Australia 6847, Australia; e-mail
kcroft@cyllene.uwa.edu.au)
Arachidonic acid can be metabolized by cytochrome P450
enzymes to a range of compounds that play a central role
in the regulation of vascular tone, renal function, and
blood pressure (1, 2). In the vasculature, smooth muscle
cells produce 20-hydroxyeicosatetraenoic acid (20-HETE)
as a major product of CYP450 metabolism. 20-HETE can
cause vasoconstriction by inhibition of potassium chan-
nels and is thought to contribute to the vasoconstrictor
action of hormones such as angiotensin II and endothelin
(3, 4). Despite the physiologic importance of CYP450
metabolites of arachidonic acid, very little is known about
the regulation of the concentration of 20-HETE in biolog-
ical fluids or the relationship of these concentrations with
physiologic state in healthy individuals. This has in part
been attributable to the lack of reliable sensitive and
specific assays to measure endogenous concentrations of
these compounds. Gas chromatography–mass spectrom-
etry (GCMS) has been used successfully to measure
20-HETE in biological samples. However, available meth-
ods rely on one or more thin-layer chromatography steps
(5, 6), and for human urine the presence of interfering
peaks can be a problem (7). An alternative procedure has
recently been reported that uses a sensitive fluorescent
HPLC assay (8 ), although this may lack the specificity of
MS.
We have developed a simplified and reliable method
for the analysis of urinary 20-HETE and analyzed 20-
HETE concentrations in 24-h urine samples from a group
of 30 healthy individuals. Our method involves the use of
a single solid-phase extraction cartridge containing both
reversed-phase and strong anion-exchange packing fol-
lowed by HPLC separation before derivatization and
GCMS analysis. We have found that preparation of the
tert-butyldimethylsilyl derivative (tBDMS), as originally
used by Prakash et al. (6), gives better chromatographic
separation from interfering peaks present in urine.
20,20-[
2
H
2
]-20-HETE was prepared according to previ-
ously published procedures (9). Unlabeled 20-HETE was
purchased from Cayman Chemicals.
␤
-Glucuronidase
(Escherichia coli), pentafluorobenzyl bromide (PFB Br),
N,N-diisopropylethylamine, and tert-butyldimethylsilyl-
N-methyltrifluoroacetamide were purchased from Sigma-
Aldrich. Pyridine was purchased from Fluka. Bond Elut-
Certify II (200 mg, 3 mL) columns were purchased from
Varian Inc. Volunteers were recruited from the general
population. We monitored 24-h blood pressures (BP) by
use of an ambulatory device (Spacelabs 90207). All studies
with samples from humans were approved by the Human
Ethics Committee of Royal Perth Hospital.
The internal standard [
2
H
2
]-20-HETE (2 ng) was added
to urine (2 mL). Each sample was left at room temperature
for 10 min to equilibrate before incubation with 0.2 mg of
␤
-glucuronidase from E. coli (in 0.075 mol/L potassium
phosphate buffer, pH 6.8, containing 1 g/L bovine serum
albumin) for2hat37°C. After hydrolysis, samples were
diluted with 2 mL of 0.1 mol/L sodium acetate solution
(pH 7) containing 50 mL/L methanol, and the pH was
adjusted to 6.0 with 100 mL/L acetic acid.
Bond Elut-Certify II columns were preconditioned with
2 mL of methanol, followed by 2 mL of 0.1 mol/L sodium
acetate solution (pH 7) containing 50 mL/L methanol
before application of the urine samples. The columns
were washed with 2 mL of methanol–water (1:1 by
volume), and urinary 20-HETE and internal standard
were eluted with 2 mL of hexane–ethyl acetate (75:25 by
volume) containing 10 mL/L acetic acid. The organic
224 Technical Briefs

extracts were evaporated to dryness under reduced pres-
sure and reconstituted in 50
␮
L of methanol for HPLC
purification on an Agilent 1100 system. Separations were
carried out with a LiChrospher
®
RP-18 (length, 100 mm; 5
␮
m bead size; Agilent) column with a linear gradient
mobile phase starting from acetonitrile–water–acetic acid
(50:50:0.05 by volume) to acetonitrile at a flow rate of 1
mL/min for 20 min. 20-HETE and the internal standard
eluted at a retention time of 6.9 min, and 1-min fractions
were collected between 6.5 and 7.5 min with an auto-
mated fraction collector (Gilson).
Fractions containing the HETEs were dried under re-
duced pressure, and the residue was treated with 40
␮
Lof
100 g/L PFB Br in acetonitrile and 20
␮
L of 100 g/L
N,N-diisopropylethylamine in acetonitrile for 30 min at
room temperature. After drying under nitrogen, the resi-
dues were treated with 20
␮
LofN-(tert-butyldimethylsi-
lyl)-N-methyltrifluoroacetamide and 10
␮
L of pyridine for
20 min at 45 °C. Samples were dried under nitrogen and
dissolved in 30
␮
L of isooctane for analysis on an Agilent
5973 GCMS. Samples (1
␮
L) were injected on a HP-1MS
column [15 m ⫻ 0.25 mm (i.d); 0.25-
␮
m film thickness;
Agilent] with a temperature program of 160 °C initially
held for 0.50 min and increased to 300 °C at a rate of
15 °C/min. Helium was used as the carrier gas, and
injections were made in the splitless mode. The mass
spectrophotometer was operated in the negative chemical
ionization mode with methane reagent gas at a source
pressure of 2 ⫻ 10
⫺4
Torr. For selected-ion monitoring
analyses, the m/z 433 (endogenous 20-HETE-PFB-tBDMS)
and m/z 435 ([
2
H
2
]-20-HETE-PFB-tBDMS) ions were mon-
itored. Urinary 20-HETEs were identified by comparison
against the retention time of an authentic 20-HETE stan-
dard, and concentrations were determined by peak-area
ratios of the analyte to internal standard [
2
H
2
]-20-HETE.
This highly sensitive and specific assay for 20-HETE in
human urine uses a dideuterated autologous 20-HETE as
internal standard for GCMS analysis. As reported previ-
ously (6 ), the tBDMS-PFB derivative has good chromato-
graphic properties, with major negative ion fragments at
m/z 433 (M-PBF) for 20-HETE and m/z 435 for
2
H
2
-20-
HETE. Fig. 1 shows a typical selected-ion chromatogram
obtained from a human urine sample. Although most
urine samples show a range of peaks in the region of
20-HETE, using this purification system and the tBDMS
derivative we were able to clearly detect 20-HETE in each
of ⬎30 individual urine samples analyzed. However,
when we used the trimethylsilyl derivative, we were not
able to clearly determine 20-HETE because of contaminat-
ing peaks. The limit of detection for this assay was ⬍0.2
pg injected on the column (signal-to-noise ratio ⫽ 28), and
the assay has good intraassay reproducibility (CV ⫽ 5%;
n ⫽ 10) for a urine sample containing 264 pmol/L
20-HETE. The interassay variation for the same urine
sample over 10 assays was 10%.
We examined urinary excretion of 20-HETE in 30
healthy individuals. For the 24-h urine samples (Table 1;
results expressed as either pmol/L or pmol/24 h). All
samples were treated with glucuronidase because it has
previously been shown that most 20-HETE in urine exists
as the glucuronide (6). There is little information available
on 20-HETE excretion in normal human urine. Prakash et
al. (6 ) studied four healthy individuals with a mean
20-HETE concentration of ⬃400 ng/L (⬃1250 pmol/L).
Although our mean value was somewhat lower than this,
Table 1. 20-HETE excretion in 30 healthy adult volunteers.
Mean (SD) age, years 55 (2)
Mean (SD) body mass index, kg/m
2
26.4 (0.6)
Gender, M/F 10/20
Mean (SD) 24-h systolic BP, mmHg 115.4 (1.2)
Mean (SD) 24-h diastolic BP, mmHg 69.5 (0.9)
20-HETE,
a
pmol/L
Geometric mean 206
95% confidence interval 155–272
Minimum 84
Maximum 1241
20-HETE,
a
pmol/24 h
Geometric mean 430
95% confidence interval 333–554
Minimum 143
Maximum 1703
a
20-HETE excretion was not normally distributed.
Fig. 1. Selected ion chromatograms from a human urine sample.
(Top panel), m/z 433 ion for endogenous 20-HETE. Retention of 10.15 min
corresponds exactly with an authentic 20-HETE standard. (Bottom panel), m/z
435 ion corresponding to the deuterium-labeled internal standard (2 ng).
Clinical Chemistry 50, No. 1, 2004 225

we did have some individuals falling within that range
(Table 1). Sacerdoti et al. (7) studied eight healthy indi-
viduals with 20-HETE expressed as the excretion rate (1.6
ng/min). It is difficult to compare these data with our
own because these individuals were receiving an infusion
of aminohippurate in 50 g/L dextrose in water at 1.5
mL/min for5htodetermine renal plasma flow.
Information on the physiologic effects of 20-HETE in
humans is limited. Sacerdoti et al. (7) showed that the rate
of 20-HETE excretion was increased in individuals with
hepatic cirrhosis. Laffer et al. (5) recently showed a
positive correlation between diastolic BP and 20-HETE
excretion rate in a group of 13 salt-sensitive hypertensive
individuals. In some rat models of hypertension, high BP
has been associated with increased 20-HETE production
(2), but this is yet to be confirmed in human studies.
In conclusion, the sample preparation procedure using
solid-phase cartridge extraction and HPLC purification
would lend itself to automation and enable the convenient
analysis of 20-HETE excretion in large human studies.
Such studies could examine in more detail the potential
role of this vasoactive CYP450 metabolite in vascular
function and human hypertension.
This study was funded in part by grants from the Na-
tional Health and Medical Research Council of Australia
(NHMRC; Grant 139067) and NIH Grant GM31278 (to
J.R.F.).
References
1. Roman RJ. P-450 metabolites of arachidonic acid in the control of cardiovas-
cular function. Physiol Rev 2002;82:131– 85.
2. McGiff JC, Quilley J. 20-Hydroxyeicosatetraenoic acid and epoxyeicosatrienoic
acids and blood pressure. Curr Opin Nephrol Hypertens 2001;10:231–7.
3. Croft KD, McGiff JC, Sanchez-Mendoza A, Carroll MA. Angiotensin II releases
20-HETE from rat renal microvessels. Am J Physiol Renal Physiol 2000;279:
F544 –51.
4. Harder DR, Campbell WB, Roman RJ. Role of cytochrome P-450 enzymes and
metabolites of arachidonic acid in the control of vascular tone. J Vasc Res
1995;32:79 –92.
5. Laffer CL, Laniado-Schwartzman M, Wang MH, Nasjletti A, Elijovich F.
Differential regulation of natriuresis by 20-hydroxyeicosatetraenoic acid in
human salt-sensitive versus salt-resistant hypertension. Circulation 2003;
107:574 –8.
6. Prakash C, Zhang JY, Falck JR, Chauhan K, Blair IA. 20-Hydroxyeicosatetra-
enoic acid is excreted as a glucuronide conjugate in human urine. Biochem
Biophys Res Commun 1992;185:728 –33.
7. Sacerdoti D, Balazy M, Angeli P, Gatta A, McGiff JC. Eicosanoid excretion in
hepatic cirrhosis. Predominance of 20-HETE. J Clin Invest 1997;100:1264 –
70.
8. Maier KG, Henderson L, Narayanan J, Alonso-Galicia M, Falck JR, Roman RJ.
Fluorescent HPLC assay for 20-HETE and other P-450 metabolites of arachi-
donic acid. Am J Physiol Heart Circ Physiol 2000;279:H863–71.
9. Lin F, Rios A, Falck JR, Belosludtsev Y, Schwartzman ML. 20-Hydroxyeicosa-
tetraenoic acid is formed in response to EGF and is a mitogen in rat proximal
tubule. Am J Physiol 1995;269:F806 –16.
DOI: 10.1373/clinchem.2003.025775
Rapid Genotyping for Tumor Necrosis Factor-
␣
(TNF-
␣
)
ⴚ863C/A Promoter Polymorphism That Determines
TNF-
␣
Response, Michael Heesen,
1*
Dagmar Kunz,
2
Mar-
tina Wessiepe,
3
Tom van der Poll,
4
Aeilko H. Zwinderman,
5
and Brunhilde Blomeke
6
(Departments of
1
Anesthesia,
4
Ex-
perimental Internal Medicine, and
5
Clinical Epidemiol-
ogy and Biostatistics, Academic Medical Center, Univer-
sity of Amsterdam, Amsterdam, The Netherlands;
Departments of
2
Clinical Chemistry and Pathobiochem-
istry,
3
Transfusion Medicine, and
6
Dermatology, Univer-
sity Hospital, Aachen, Germany; * address correspon-
dence to this author at: Department of Anesthesia,
Academic Medical Center, University of Amsterdam,
Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands;
fax 31-20-6979441, e-mail m.heesen@amc.uva.nl)
Tumor necrosis factor-
␣
(TNF-
␣
) plays a central role in
orchestrating the inflammatory response (1). Accord-
ingly, blocking TNF-
␣
activity has become a standard
treatment of several inflammatory diseases (2, 3). TNF-
␣
production shows high interindividual variations, which
have been assigned mainly to inherited factors (4). Sev-
eral genetic polymorphisms related to TNF-
␣
synthesis
have been detected in the TNF gene (5, 6). The ⫺308
promoter polymorphism was found to affect TNF-
␣
pro-
duction by some authors (7 ) but not by others (8). Similar
inconsistencies have been found for the association of this
polymorphic site with susceptibility to and/or outcome of
sepsis (8, 9). The NcoI polymorphism located within the
first intron of the lymphotoxin A (LTA) gene was reported
to be associated with TNF-
␣
plasma concentrations (8 ).In
a recent report, de Jong et al. (10) found no relationship
between ex vivo TNF-
␣
production on endotoxin stimu-
lation of human whole blood and ⫹489, ⫺238, and ⫺376
single-nucleotide polymorphisms or TNFa microsatellites
of the TNF-
␣
gene (10). Thus, the genetic factors deter-
mining the TNF-
␣
response to infection are still poorly
defined.
Recently, Skoog et al. (11 ) identified a C/A exchange at
position ⫺863 of the TNF-
␣
gene promoter and found
higher transcriptional activity of the C allele in reporter
gene assays. This polymorphic site was found to be
associated with thyroid-associated ophthalmopathy (12 ),
Crohn disease (13), juvenile rheumatoid arthritis (14 ),
and the lumbar spine area (15 ).
In the present study we sought to determine the asso-
ciation of the ⫺863 TNF-
␣
promoter polymorphism with
the TNF-
␣
production capacity of human blood cells. We
describe a new real-time PCR assay with specific fluores-
cently labeled hybridization probes for genotyping for
this polymorphism. We also genotyped samples for other
polymorphic sites implicated in TNF-
␣
production capac-
ity and evaluated the possible existence of linkage dis-
equilibria with the ⫺863 polymorphism under study.
Unrelated, nonsmoking, male blood donors of Cauca-
sian origin (age range, 18 –65 years) were included. The
period of inclusion was 2 weeks. Exclusion criteria in-
cluded acute or chronic diseases or any medication within
3 weeks before being enrolled into this study. This study
226 Technical Briefs